CN106596532B - A kind of detection method of simple alkaline phosphatase activities - Google Patents
A kind of detection method of simple alkaline phosphatase activities Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 38
- 230000000694 effects Effects 0.000 title claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
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- 239000008107 starch Substances 0.000 claims abstract description 12
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940005657 pyrophosphoric acid Drugs 0.000 claims abstract description 10
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000010148 water-pollination Effects 0.000 claims abstract description 6
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims abstract description 5
- 102100022624 Glucoamylase Human genes 0.000 claims abstract description 5
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- 239000003795 chemical substances by application Substances 0.000 claims description 15
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- 238000005259 measurement Methods 0.000 claims description 9
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- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 4
- 235000011180 diphosphates Nutrition 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
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- 230000002596 correlated effect Effects 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 2
- -1 phosphate anion Chemical class 0.000 claims description 2
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- 238000000034 method Methods 0.000 abstract description 7
- 230000007062 hydrolysis Effects 0.000 abstract description 4
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 4
- 239000000523 sample Substances 0.000 abstract description 4
- 238000005903 acid hydrolysis reaction Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000007864 aqueous solution Substances 0.000 description 7
- 239000004033 plastic Substances 0.000 description 6
- 229920003023 plastic Polymers 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
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- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 description 2
- 238000003698 laser cutting Methods 0.000 description 2
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- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 206010056375 Bile duct obstruction Diseases 0.000 description 1
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 1
- 206010004659 Biliary cirrhosis Diseases 0.000 description 1
- 206010008609 Cholangitis sclerosing Diseases 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 102000002568 Multienzyme Complexes Human genes 0.000 description 1
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- 238000012742 biochemical analysis Methods 0.000 description 1
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- 208000020694 gallbladder disease Diseases 0.000 description 1
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- 239000007788 liquid Substances 0.000 description 1
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- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 201000000742 primary sclerosing cholangitis Diseases 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
The invention discloses a kind of detection methods of simple alkaline phosphatase activities.Using the alkaline phosphatase analyte in sample solution after being catalyzed pyrophosphoric acid hydrolysis, Cu2+Due to not being complexed by pyrophosphoric acid can high efficiency inhibit diastatic activity so that water-soluble lower starch is not by Glucoamylase hydrolysis.Starch in reaction solution makes corresponding sheet occur to be changed into hydrophobic wettability modification from hydrophily after being added dropwise in paper microfluidic analysis device, and then the volume for making detection reagent solution can flow through the region reduces, and it is shorter to eventually lead to its length of flow in paper device.The length of flow of detection reagent solution is inversely proportional to the active size of sample alkaline phosphatase in paper device, that is, completes the quantitative detection of alkaline phosphatase activities.The method of the present invention is simple and easy, and the operator without professional training can also carry out experiment, and carry out quantifiable signal reading without using any instrument, greatly reduction analysis cost, has broad application prospects.
Description
Technical field
The invention belongs to biochemical analysis and biosensor technique field, and in particular to a kind of alkaline phosphatase of simple low cost
The detection method of enzymatic activity.
Background technique
Alkaline phosphatase, also referred to as alkaline phosphatase, can catalytic phosphatase ester bond under alkaline condition hydrolysis.Lactation is dynamic
In object, the alkaline phosphatase activities in liver, bile duct, kidney, bone and placenta are relatively high.Common alkaline phosphatase includes intestines
Road alkaline phosphatase, non-tissue specificity alkaline phosphatase and P-ALP.Alkaline phosphatase activities are too high or too low
It is related to a variety of diseases.For example, the activity of the colon cancer cell alkaline phosphatase of differentiation can be increased significantly, it is taken as colon cancer
The qualitative and quantitative index of cell differentiation.The raising of serum neutral and alkali phosphate is referred to as high-alkali acid phosphatase mass formed by blood stasis,
It is considered and the liver and gallbladder diseases such as pernicious bile duct obstruction, Biliary Cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and hepatosarcoma
Disease is closely related.Serum activity change of Alkaline phosphatase is too low also related to some diseases.Therefore, biological sample alkaline phosphatase
Active quantitative detection has highly important medical significance.Current existing alkaline phosphatase activities detection method mainly by
The analysis instruments such as microplate reader, luminoscope, electrochemical workstation realize quantitative detection.However, these methods require operator
Higher professional skill, especially used instrument price is costly and bulky, leads to global analysis higher cost.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of alkaline phosphatase activities of simple low cost
Detection method.
Thinking of the invention: carrying out various biochemical reactions in the solution and quantitative letter is carried out on paper microfluidic analysis device
Number read.The biochemical reaction carried out in solution includes that alkaline phosphatase specific catalytic pyrophosphoric acid hydrolysis, pyrophosphoric acid are special
Bivalent cupric ion (Cu is complexed2+) reaction, Cu2+Inhibit diastatic activity reaction and carbohydrase specific catalytic water solubility lower
Soluble starch hydrolysis generate water-soluble good glucose response.End reaction solution drop containing soluble starch molecule
It is incited somebody to action after being added in paper device so that corresponding sheet occurs to be changed into hydrophobic wettability modification from hydrophily, and keeps hydrophily color
Color detection reagent solution flows through the length of flow reduction behind the region based on capillarity.Hydrophily colour detection reagent solution
Length of flow in paper device is inversely proportional to the hydrophilic-hydrophobic wettability modification degree of sheet, and the latter is inversely proportional to sample
The active size of alkaline phosphatase.In other words, by simply measuring in paper device colored detection reagent solution in paper device
Length of flow can carry out the quantitative detections of alkaline phosphatase activities.Therefore, enzyme is replaced by using the extremely low ruler of price
The analysis instruments such as instrument, luminoscope, electrochemical workstation are marked, or directly reads the included length scales scale of paper device and carries out signal
It reads, analysis cost can be significantly reduced.
Specific steps are as follows:
Step 1 mixes alkaline phosphatase sample solution with pyrophosphate solution, and pyrophosphoric acid molecule is by alkaline phosphatase spy
The opposite sex is hydrolyzed to phosphate anion, and Cu is then added2+Solution carries out Cu2+Pyrophosphoric acid complex reaction, adds Glucoamylase Solution
Carry out Cu2+Inhibit diastatic activity reaction, soluble starch solution is then added and carries out saccharification enzymatic hydrolysis starch reaction, system
Obtain end reaction solution.
End reaction solution made from step 1 is added drop-wise to the round reaction solution of paper microfluidic analysis device by step 2
Area is added, and then color agents solution, the color agents are added dropwise in the circle detection reagent of paper microfluidic analysis device addition area
Solution will flow through round reaction solution addition area and enter rectangle measurement area.
Step 3 measures it in rectangle and measures area after color agents solution stops flowing in rectangle measurement area
In length of flow, the size of the length and the size of alkaline phosphatase sample solution activity change of Alkaline phosphatase are negatively correlated,
Complete the quantitative detection of alkaline phosphatase activities.
The paper microfluidic analysis device is prepared by hydrophily paper, containing continuously and according to the addition of circle detection reagent
Area, round reaction solution addition area and rectangle measure the tactic three parts functional area in area.
The color agents are one of water-soluble color inks, coloured dye and coloured pigments.
The rectangle measures area and is provided with length scales or not set length scales, when being provided with length scales,
Color agents solution can be directly read and measure the length of flow in area in rectangle, when not set length scales, using straight
Ruler measurement color agents solution measures the length of flow in area in rectangle.
Compared with existing alkaline phosphatase activities detection method, of the invention is had the prominent advantages that: 1) analytic process letter
Single easy, the operator without professional training can also carry out experiment；2) using the extremely low ruler of price or to directly read paper micro-
The included length scales scale of flow control analytical equipment carries out signal-obtaining, can greatly reduce analysis cost；3) it can directly promote and answer
Active level for various body fluid sample alkaline phosphatase analytes detects, and has in fields such as clinical diagnosis, medical researches
Have broad application prospects.
Detailed description of the invention
Fig. 1 is the detection method of the alkaline phosphatase activities of the simple low cost in Example 1 and Example 2 of the present invention
Schematic illustration.
Marked in the figure: 1-test tubes；2-buffer solutions；3-alkaline phosphatases；4-pyrophosphoric acids；5-phosphate radicals；
6—Cu2+；7-carbohydrase；7-1—Cu2+Be saccharified multienzyme complex；8-soluble starches；9-glucose；10-not set length
The paper microfluidic analysis device of anale settting scale；10-1-circle detection reagent adds area；10-2-circle reaction solution adds area；
10-3-rectangle measures area；11-the traces formed by reaction solution；12-color agents solution；13-color agents solution
Flowing trace in paper microfluidic analysis device；14-plastic flat rulers.
Fig. 2 is that the detection method of the alkaline phosphatase activities in the embodiment of the present invention 1 using simple low cost detects 5U/mL
The length of flow of red ink aqueous solution obtained by alkaline phosphatase sample solution and the flowing of red ink aqueous solution obtained by blank sample are long
The comparison figure of degree.
Fig. 3 is that the detection method of the alkaline phosphatase activities in the embodiment of the present invention 2 using simple low cost detects a system
The length of flow and alkaline phosphatase activities of red ink aqueous solution obtained by the alkaline phosphatase sample solution of column different activities concentration
Log value between working curve.
Specific embodiment
Following embodiment will be further described the present invention, but not thereby limiting the invention.
Embodiment 1:
Using simple low cost alkaline phosphatase activities detection method detection 5U/mL alkaline phosphatase sample solution and
Blank sample (the not buffer solution of analyte-containing).
As shown in Figure 1, specific implementation process is as follows: 15 μ L are added in step 1 in the plastics test tubes of a 1.5mL
5U/mL alkaline phosphatase sample solution (by the 10mM tris-HCl buffer preparation of the NaCl containing 100mM, pH 7.4), with
15 μ L, 200 μM of pyrophosphate solutions is added afterwards (by the 10mM tris-HCl buffer preparation of the NaCl containing 100mM, pH 7.4)
It is mixed with, is placed at 37 DEG C and reacts 60min, continuously add 15 μ L, 50 μM of copper nitrate aqueous solutions, react 15min under room temperature,
It is further continued for being added 15 μ L 1mg/mL Glucoamylase Solutions (being prepared by 20mM acetic acid-sodium acetate buffer solution, pH 4.5), under room temperature
70min is reacted, 15 μ L, 2% soluble starch solution is then added (after being heated by the ultrapure water that resistivity is 18.2M Ω cm
Prepare), 2h is reacted at 40 DEG C, and end reaction solution is made；Step 2 pipettes 5.6 μ L end reaction solution and is added drop-wise to paper miniflow
Analytical equipment is controlled (by CO2Laser cutting instrument cutting double-round board qualitative filter paper is prepared；Laser cutting electric current and speed are respectively
5mA and 20mm/s) round reaction solution add area (diameter 2.2mm), then paper microfluidic analysis device circle inspection
Test agent adds area (diameter 2.2mm) and the heroic board red ink solution that 5.5 μ L dilute 20 times through ultrapure water, the red ink is added dropwise
Solution will flow through round reaction solution addition area and enter rectangle measurement area；Step 3, when red ink solution is measured in rectangle
After stopping flowing in area's (length and width is respectively 15 and 0.9mm), the flowing for measuring the reagent using common plastics ruler is long
Degree, the size of the length and the size of alkaline phosphatase sample solution activity change of Alkaline phosphatase are negatively correlated, that is, complete alkalinity
The quantitative detection of phosphatase activity.
According to identical step, analysis margin sample, i.e. 10mM tris-HCl buffer solution (NaCl containing 100mM, pH
7.4), and red ink solution is measured using common plastics ruler measure the length of flow in area, i.e. space length value in rectangle.
Figure it is seen that detection 5U/mL alkaline phosphatase sample solution is resulting compared with detecting the resulting length value of blank sample
Length value almost can be ignored.This is because the alkaline phosphatase in sample solution is after being catalyzed pyrophosphoric acid hydrolysis,
Complexing Cu is removed almost without free pyrophosphoric acid2+, so that the metal ion can inhibit diastatic activity with high efficiency, thus solvable
Property starch molecule is effectively maintained.These water-soluble lower molecules are very big after being added dropwise in paper microfluidic analysis device
The hydrophobicity for increasing corresponding sheet causes to flow to rectangle detection zone almost without red ink solution.Comparative experiments in Fig. 2
The result shows that the detection method of the alkaline phosphatase activities of simple low cost is practical.
Embodiment 2:
Detection method detection active concentration range using the alkaline phosphatase activities of simple low cost is 0.0375~5U/
The alkaline phosphatase sample solution of mL.
As shown in Figure 1, detecting the specific steps of each alkaline phosphatase sample solution in the present embodiment are as follows: step 1,
The alkaline phosphatase sample solution of the 15 a certain active concentrations of μ L is added in the plastics test tubes of one 1.5mL (by containing 100mM
The 10mM tris-HCl buffer preparation of NaCl, pH 7.4), 15 μ L, 200 μM of pyrophosphate solutions are then added (by containing
The 10mM tris-HCl buffer preparation of 100mM NaCl, pH 7.4) it is mixed with, it is placed at 37 DEG C and reacts 60min,
15 μ L, 50 μM of copper nitrate aqueous solutions are continuously added, react 15min under room temperature, are further continued for that 15 μ L 1mg/mL Glucoamylase Solutions are added
(being prepared by 20mM acetic acid-sodium acetate buffer solution, pH 4.5) reacts 70min under room temperature, it is soluble that 15 μ L 2% is then added
Starch solution (is prepared) after being heated by the ultrapure water that resistivity is 18.2M Ω cm, reacts 2h at 40 DEG C, it is molten that end reaction is made
Liquid；Step 2 pipettes 5.6 μ L end reaction solution and is added drop-wise to paper microfluidic analysis device (by CO2It is cut by laser instrument and cuts double-round
Board qualitative filter paper is prepared；Being cut by laser electric current and speed is respectively 5mA and 20mm/s) round reaction solution add area
Then 5.5 μ are added dropwise in circle detection reagent addition area (diameter 2.2mm) of paper microfluidic analysis device in (diameter 2.2mm)
L dilutes 20 times of heroic board red ink solution through ultrapure water, which will flow through round reaction solution addition area and enter
Rectangle measures area；Step 3 is stopped when red ink solution measures in area (length and width is respectively 15 and 0.9mm) in rectangle
After fluid stopping is dynamic, the length of flow of the reagent is measured using common plastics ruler.All alkaline phosphatase sample solutions are obtained
The length of flow of red ink aqueous solution maps (Fig. 3) to the Log value of alkaline phosphatase activities concentration, that is, completes to be based on length of flow
The quantitative detection of the alkaline phosphatase activities of measurement.
From the figure 3, it may be seen that with the reduction of alkaline phosphatase activities concentration, the length of flow of corresponding red ink aqueous solution by
It is cumulative big.This is because hydrolyzed pyrophosphoric acid does not retain then relatively multi-purpose when sample solution activity change of Alkaline phosphatase is lower
In the more Cu of complexing2+.And with greater activity carbohydrase, then a large amount of water-soluble bad soluble starch of catalyzing hydrolysis divides
Son.The wetability for changing corresponding sheet after being added dropwise in paper device containing water-soluble good glucose products is smaller, so that
More red ink solution can flow to rectangle detection zone, generate longer length of flow.In addition, Fig. 3 is shown, this reality is utilized
The method for applying example measures the Log value of resulting length of flow value and alkaline phosphatase activities concentration in 0.0375~5U/mL range
It is interior that good linear relationship is presented.
Claims (1)
1. a kind of detection method of simple alkaline phosphatase activities, it is characterised in that specific steps are as follows:
Step 1 mixes alkaline phosphatase sample solution with pyrophosphate solution, and pyrophosphoric acid molecule is by alkaline phosphatase enzyme spcificity
It is hydrolyzed to phosphate anion, Cu is then added2+Solution carries out Cu2+Pyrophosphoric acid complex reaction adds Glucoamylase Solution progress
Cu2+Inhibit diastatic activity reaction, soluble starch solution is then added and carries out saccharification enzymatic hydrolysis starch reaction, is made most
End reaction solution；
Step 2 adds the round reaction solution that end reaction solution made from step 1 is added drop-wise to paper microfluidic analysis device
Area, and then color agents solution, the color agents solution is added dropwise in the circle detection reagent of paper microfluidic analysis device addition area
Round reaction solution addition area will be flowed through and enter rectangle measurement area；
Step 3 measures it and measures in area in rectangle after color agents solution stops flowing in rectangle measurement area
Length of flow, the size of the length and the size of alkaline phosphatase sample solution activity change of Alkaline phosphatase are negatively correlated, i.e., complete
At the quantitative detection of alkaline phosphatase activities；
The paper microfluidic analysis device is prepared by hydrophily paper, containing it is continuous and according to circle detection reagent addition area,
Round reaction solution addition area and rectangle measure the tactic three parts functional area in area；
The color agents are one of water-soluble color inks, coloured dye and coloured pigments；
Rectangle measurement area is provided with length scales or not set length scales can when being provided with length scales
It directly reads length of flow of the color agents solution in rectangle measurement area and uses ruler amount when not set length scales
It surveys color agents solution and measures the length of flow in area in rectangle.
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| CN108088841A (en) * | 2017-12-15 | 2018-05-29 | 长春理工大学 | Paper substrate micro-fluidic chip that is a kind of while detecting four kinds of liver function enzymes and preparation method thereof |
| CN110361440B (en) * | 2018-04-09 | 2021-08-06 | 上海交通大学 | Portable electrophoresis titration detection method for activity of alkaline phosphatase |
| CN108896506B (en) * | 2018-07-16 | 2020-10-27 | 济南大学 | Method for detecting alkaline phosphatase activity and concentration of alkaline phosphatase inhibitor |
| CN109557060B (en) * | 2018-11-28 | 2020-10-20 | 广西师范大学 | Based on NH2Method for visually detecting alkaline phosphatase activity in serum by using (E) -Cu-MOF |
| CN110596085B (en) * | 2019-09-03 | 2021-01-15 | 中山大学 | Distance measurement-based non-consumable paper chip and preparation method and application thereof |
| CN110672863A (en) * | 2019-09-29 | 2020-01-10 | 桂林理工大学 | Instrument-free quantitative detection method for divalent lead ions |
| CN112877382B (en) * | 2021-01-18 | 2023-02-21 | 江南大学 | Method for preparing phosphate starch by enzyme method |
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Application publication date: 20170426 Assignee: GUILIN VEIRUN MEDICAL TECHNOLOGY Co.,Ltd. Assignor: GUILIN University OF TECHNOLOGY Contract record no.: X2023980046003 Denomination of invention: A simple method for detecting alkaline phosphatase activity Granted publication date: 20190723 License type: Common License Record date: 20231108 |